International Journal of Molecular Zoology 2024, Vol.14, No.4, 244-254 http://animalscipublisher.com/index.php/ijmz 245 researchers, clinicians, and policymakers working towards the advancement of xenotransplantation technologies and the development of regulatory frameworks to ensure the safety and efficacy of these innovative medical procedures. 2 Background on Xenotransplantation 2.1 Definition and history of xenotransplantation Xenotransplantation refers to the transplantation of living cells, tissues, or organs from one species to another. Historically, the concept of xenotransplantation has been explored as a potential solution to the shortage of human organs available for transplantation. Early attempts in the 20th century involved the use of primate organs, but these efforts were largely unsuccessful due to severe immune rejection and ethical concerns. The advent of genetic engineering has renewed interest in xenotransplantation, particularly using pigs as donors due to their anatomical and physiological similarities to humans (Wolf et al., 2019; Xi et al., 2023). 2.2 Current status of pig-to-human organ transplantation Recent advancements in genetic engineering have made pigs a viable source of organs for human transplantation. Genetically modified pigs, specifically those with knockouts of the alpha-1,3-galactosyltransferase gene, have shown promise in reducing hyperacute rejection, a major barrier in xenotransplantation (Lei et al., 2022). Clinical studies have demonstrated that kidneys from these genetically modified pigs can function in human recipients for short periods without signs of hyperacute rejection (Montgomery et al., 2022). Additionally, ongoing research focuses on further genetic modifications to address other immune responses and physiological incompatibilities, paving the way for future clinical trials (Kemter et al., 2020). 2.3 Risks associated with pathogens in xenotransplantation One of the significant risks in xenotransplantation is the potential transmission of zoonotic pathogens from donor pigs to human recipients. Pigs can harbor various infectious agents, including porcine endogenous retroviruses (PERVs), which pose a risk to immunocompromised transplant recipients (Hartline et al., 2018; Lei et al., 2022). To mitigate these risks, genetic modifications are being employed to knock down genes related to PERVs, and highly sensitive diagnostic methods are being developed to detect and monitor infectious agents in donor pigs and transplant recipients (Hartline et al., 2018). Ensuring the safety of xenotransplantation requires a comprehensive understanding of the spectrum of infectious agents in donor pigs and the implementation of stringent screening protocols. 3 Genetic Engineering Techniques 3.1 Overview of genetic engineering technologies Genetic engineering technologies have revolutionized the field of xenotransplantation, particularly in the development of pathogen-free pigs. The primary tools used in genetic engineering include CRISPR/Cas9, TALENs (Transcription Activator-Like Effector Nucleases), and ZFNs (Zinc Finger Nucleases). CRISPR/Cas9: This technology allows for precise editing of the genome by creating double-strand breaks at specific locations, which are then repaired by the cell's natural repair mechanisms. CRISPR/Cas9 has been widely used due to its simplicity, efficiency, and versatility. It has been employed to inactivate porcine endogenous retroviruses (PERVs) and to modify genes that enhance immunological compatibility with humans (Kemter et al., 2018). TALENs: These are engineered proteins that can be designed to bind to specific DNA sequences and create double-strand breaks. TALENs offer high specificity and have been used in various genetic modifications, although they are generally more complex to design and produce compared to CRISPR/Cas9. ZFNs: These are synthetic proteins that can be engineered to target specific DNA sequences. ZFNs have been used for gene editing for many years, but their use has declined with the advent of more efficient technologies like CRISPR/Cas9. 3.2 Advantages and limitations of different gene editing tools Each gene editing tool has its own set of advantages and limitations. The advantages of CRISPR/Cas9 are high efficiency, ease of design, and ability to target multiple genes simultaneously. However, when using CRISPR/Cas9, we also need to pay attention to potential off-target effects and the need for careful design to avoid unintended mutations (Kararoudi et al., 2018; Yue et al., 2020). TALENs has high specificity and lower off-target effects
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